A variety of approaches have been taken to introduce genetic material into cells of an individual. Delivery vehicles, or vectors, based on retroviruses have proven to be effective, both in the laboratory and in the clinic, for introducing genetic material into cells of an individual. Examples of such vectors include murine leukemia virus (MLV) retroviral vectors and lentiviral vectors.
MLV is an enveloped virus with an approximately 8.5 kilobase long, positively stranded RNA genome. The genome encodes two polyproteins that are proteolytically processed to form the eight individual proteins of the virus. The first polyprotein contains seven proteins: the gag region encodes matrix, p12, nucleocapsid, and capsid, which are structural proteins that package two copies of the RNA genome into the capsid. The pol region contains the protease that cleaves the polyprotein into its individual constituents, as well as the reverse transcriptase and integrase proteins important in the viral life cycle. The second polyprotein, encoded by env, is translated from mRNA that is spliced to remove gag-pol. The resulting polyprotein is proteolytically cleaved to form transmembrane (TM) and surface (SU) polypeptides that are linked by disulfide bonds to yield a transmembrane protein spanning the viral surface envelope. In addition to the coding regions, the viral genome contains several regions important for the viral life cycle, including flanking long terminal repeats (LTR) which contain the viral promoter; a region Ψ (psi) important for viral packaging; and several other elements important in viral function.
Delivery vectors based on MLV were first developed by replacing viral genes with a transgene of interest. The retroviral vectors can be deleted of all viral genes, allowing for insertion of a non-MLV gene of interest into the vector, thereby generating recombinant retroviral vectors. The gag and pol genes are supplied on a separate construct for recombinant vector production. Furthermore, env can be replaced with the surface protein of another enveloped virus, such as vesicular stomatitis virus glycoprotein (VSV-G).
Lentiviral vectors are a family of retroviruses that include the human immunodeficiency virus (HIV). Vectors based on HIV have been developed. HIV-based vectors exhibit the natural ability to infect non-dividing cells.
Retroviruses and vectors derived from them require an envelope protein in order to transduce efficiently a target cell. The envelope protein is expressed in the cell producing the virus or vector and becomes incorporated into the virus or vector particles. Use of envelope protein derived from one virus other to package a different virus is known as pseudotyping. As noted above, the endogenous envelope protein of MLV is sometimes replaced with VSV-G, which exhibits greater stability than the MLV envelope protein and can better withstand the conditions of viral purification. In addition, VSV-G allows retroviral vectors to deliver genes to a broad range of target cell types.
There is continued interest in developing pseudotyped retroviral vectors, e.g., pseudotyped recombinant retroviral vectors.
Literature
U.S. Pat. No. 5,817,491; U.S. Pat. No. 5,739,018; U.S. Pat. No. 6,133,027; U.S. Pat. No. 6,432,705; Parrott et al. (2003) Mol. Ther. 8:688-700; Schlehuber and Rose (2004) J. Virol. 78:5079-5087; Azzouz et al. (2004)Nature 429:413-417; Guibing a et al. (2004) Molec. Therapy 9:76-84; Wong et al. (2004) Molec. Therapy 9:101-111; Li et al. (2003) J. Virol. 67:4070-4077.